DESCRIPTION: The decision to initiate DNA replication is carefully controlled during the cell cycle, and uncontrolled replication is a distinguishing feature of neoplasia. This proposal will investigate molecular mechanisms that control replication in eukaryotic cells. Specific Aims 1 and 2 will determine the precise nature of the changes at autonomously replicating sequences (ARS's), using double-and single- strand specific chemical and nucleolytic probes in combination with genomic footprint, supercoiling, and replication bubble assays; determine, using site-directed mutagenesis, which of the known cis- acting elements in ARS l are required for this same step in replication to occur; and identify proteins that trigger or participate in this step, using conditional mutations in proteins known to act at replication origins. These studies will define a specific intermediate in the initiation of replication in vivo, and provide critical information to workers attempting to recapitulate replication reactions in vitro. Specific Aim 3 will determine which step(s) in the assembly and activation of replication initiation complexes is inhibited in an ARS that is active in plasmids but not in the chromosome. This study thus will define another point at which initiation of DNA replication is regulated. Finally, studies in the investigator's laboratory have identified two novel proteins (p24 and p25) that are essential for cell viability and interact with Orc2p(a known ARS-associated protein). Conditional mutations in p24 and p25 will be isolated in Specific Aim 4 to determine the role these proteins play in linking initiation complexes to the assembly of replication forks in vivo.